Calcium determination in serum with stable alkaline Arsenazo III and triglyceride clearing

Abstract

We describe an analytical procedure for determining serum calcium, using the ligand Arsenazo III in an aqueous alkaline medium. The choice of pH for the proposed technique differs from current procedures, which are for the most part carried out in a slightly acidic medium. An acidic medium avoids interference from magnesium, but is spectrophotometrically suboptimal for this pH-dependent reaction: the molar absorptivity of the Arsenazo III complex with calcium at acid pH is 13 787 L mol-1 cm-1, about one-half of that at a more optimal alkaline pH (26574 L mol-1 cm-1). We have included a clearing technique in the reagent to avoid spectral aberrations from hypertriglyceridemic samples. α-Cyclodextrin absorbs the nonesterified fatty acids liberated from triglycerides by a microbial lipase. This modification may also be helpful for binding nonesterified fatty acids, which are known to interfere with calcium procedures by forming calcium soaps and thus preventing the reaction with intended ligands. The use of 8-hydroxyquinoline sulfonate as the magnesium-masking agent facilitates the use of alkaline pH. The less-water-soluble alternative, 8-hydroxyquinoline, commonly used as a precipitating agent for several methods, is difficult to solubilize in the alkaline reagent and tends to precipitate when complexed to magnesium. Finally, the use of alkaline pH results in a prolonged (≥6 weeks) shelf life for the reagent.